Changes in Intracellular Ca²⁺ Induced by GABA_A Receptor Activation and Reduction in Cl⁻ Gradient in Neonatal Rat Neocortex

Abstract

We have studied the effects of gamma-aminobutyric acid (GABA) and of reducing the Cl- gradient on the [Ca2+]i in pyramidal neurons of rat somatosensory cortex. The Cl- gradient was reduced either with furosemide or by oxygen-glucose deprivation. Immature slices taken at postnatal day (P)7-14 were labeled with fura-2, and [Ca2+]i was monitored in identified pyramidal cells in layer II/III as the ratio of fluorescence intensities (RF340/F380). The magnitude of the [Ca2+]i increases induced by oxygen-glucose deprivation was significantly reduced (by 44%) by bicuculline (10 microM), a GABAA receptor antagonist. Under normal conditions, GABA generally did not raise [Ca2+]i, although in some neurons a small and transient [Ca2+]i increase was observed. These transient [Ca2+]i increases were blocked by Ni2+ (1 mM), a blocker of voltage-dependent Ca2+ channels (VDCCs). Continuous perfusion with GABA did not cause a sustained elevation of [Ca2+]i but bicuculline caused [Ca2+]i oscillations. After inhibition of Cl- extrusion with furosemide (1.5 mM), GABA induced a large [Ca2+]i increase consisting of an initial peak followed by a sustained phase. Both the initial and the sustained phases were eliminated by bicuculline (10 microM). The initial but not the sustained phase was abolished by Ni2+. In the presence of Ni2+, the remaining sustained response was inhibited by the addition of 2-amino-5-phosphonopentanoic acid (AP5, 20 microM), a selective N-methyl-D-aspartate (NMDA) receptor antagonist. Thus the initial peak and the sustained phase of the GABA-evoked [Ca2+]i increase were mediated by Ca2+ influx through VDCCs and NMDA receptor channels, respectively, and both phases were initiated via the GABAA receptor. These results indicate that, in neocortical pyramidal neurons, a reduction in the Cl- gradient converts the GABAA receptor-mediated action from nothing or virtually nothing to a large and sustained accumulation of cellular Ca2+. This accumulation is the result of Ca2+ influx mainly through the NMDA receptor channel. Thus GABA, normally an inhibitory transmitter, may play an aggravating role in excitotoxicity if a shift in the Cl- equilibrium potential occurs, as reported previously, during cerebral ischemia.

GABAergic Input onto CA3 Hippocampal Interneurons Remains Shunting throughout Development

“…In PCs, both cotransporters are developmentally regulated (Fukuda et al, 1998;Rivera et al, 1999;Yamada et al, 2004;Chudotvorova et al, 2005;Dzhala et al, 2005) with increased KCC2 transport expression being correlated with the developmental shift from depolarizing IPSPs to hyperpolarizing IPSPs (Rivera et al, 1999;Chudotvorova et al, 2005). Although both appear to have a role in controlling SLIN Cl Ϫ homeostasis, neither KCC2 nor NKCC1 were regulated over the developmental window investigated, highlighting another important distinction between the regulation of PC and SLIN GABAergic transmission.…”

In the Adult Hippocampus, Chronic Nerve Growth Factor Deprivation Shifts GABAergic Signaling from the Hyperpolarizing to the Depolarizing Direction

“…In the developing hippocampus, the cation-chloride cotransporter NKCC1 appears to be the main Cl Ϫ uptake mechanism responsible for the depolarizing action of GABA (Rohrbough and Spitzer, 1996;Plotkin et al, 1997;Fukuda et al, 1998;Sun and Murali, 1999;Li et al, 2002;Yamada et al, 2004;Rivera et al, 2005;Sipila et al, 2006;Blaesse et al, 2009). The developmental increase in KCC2 expression, and in particular the KCC2b isoform, is responsible for the developmental increase in neuronal Cl Ϫ extrusion and for the ontogenetic shift of GABA from the depolarizing to the hyperpolarizing direction (Rivera et al, 1999;Yamada et al, 2004).…”

Temporal Development of Cyclic Nucleotide-Gated and Ca2+-Activated Cl− Currents in Isolated Mouse Olfactory Sensory Neurons

“…Several developing neurons of some brain structures, including the hippocampus, the neocortex, and the hypothalamus, have a higher intracellular concentration of Cl Ϫ than adult neurons. Intracellular Cl Ϫ accumulation in some immature neurons is caused by the Na-K-2Cl cotransporter NKCC1 (Fukuda et al 1998;Yamada et al 2004). In adult neurons K-Cl cotransporters lower intracellular Cl Ϫ below its equilibrium potential, modifying GABA action from excitatory to inhibitory.…”